General Constitutional genetics Flashcards
Question
Answer
Name 2 X-Linked Recessive disorders
- Duchenne/Becker Muscular Dystrophy (DMD/BMD)- Spinal and Bulbar Muscular Atrophy (SBMA)- Androgen Insensitivity Syndrome (AIS)
Name 2 X-Linked Dominant disorders
X-linked hypophosphatemiaX-linked Alport SyndromeRett Syndrome
Name three clinical features associated with CF
Chronic coughing and wheezingFailure to thrivePancreatic insufficiency
Name the most common CF mutation
p.Phe508del - 75%
Describe the function of CFTR (7q31.2)
Cyclic AMP-activated chloride channel located in the plasma membrane of secretory eithelial cells
Briefly describe CRISPR-Cas9
Gene editing - Cas9 protein complex containing specific sequence of RNA - once complimentary sequence is identified the DNA is cut and released into the cell. Cellular DNA repair mechanisms repair the break - but this is prone to error. By introducing templates of ‘corrected’ DNA - these specific sequences can be incorporated to replace mutant alleles
Name 3 disorders associated with FMR1
- Fragile X, 2. FXTAS,3. POI
Name 7 methods for detecting UPD
MS-PCRMS-Melt Curve AnalysisMS-MLPAMS-PyrosequencingMicrosatellite analysisSNP ArraysWGS/WES - trios especially powerful
Name 3 methods of detecting UPD that rely on bisulphite conversion
MS-PCRMS-Melt Curve AnalysisMS-Pyrosequencing
What are the pros and cons of using microsatellite analysis for UPD detection?
Pros: Will distinguish whole/partial UPD and Hetero/Isodisomy, relatively cheap, no prior conversion step, reliable methodCons: Need parental samples and informative markers
Describe MS-MLPA
2 tubes, 1 for CNV, 1 for methylation specific enzyme digest. Methylated DNA won’t digest, and then will amplify during MLPA.Semi quantitative,no parental bloods needed, can distinguish UPD from deletion from a small amount of DNAcan’t distinguish UPD and mutation in imprinting centre
How can you use WGS/WES for detection of UPD?
Trio analysis - bioinformatic pipelines will check for identity by looking for biparental inheritanceCan detect mosaics and can distinguish full range of del/UPD/IC defectExpensive and time consuming, requires parental samples
What are the pros and cons of using bisulphite conversion for UPD testing?
Pros: Allows for analysis without parental samples, bisulphite conversion kits readily available, cheap and effectiveCons: Won’t distinguish segmental/whole UPD, or del from UPD (MS-PCR)
what are antisense oligonucleotides?
ssDNA or RNA 20bp that binds to mRNA blocking the translational mechanism. The can be used to block production of abnormal protein, correct aberration by exon skipping or correct splice mutations. they are checmically modified to prevent nuclease degradation
give examples of how antisense oligos have been used to treat diseases?
- DMD - Eteplirsen (Exondys 51) in clinical use. used as reading frame correction to induce exon skipping and produce smaller partially functional protein. requires repeated treatments. it is mutation specific but due to hotspots is applicable to wide number of patients. low efficiency in heart muscle and many patients die of heart complications so need to improve AO efficiency in heart muscles - would allow lower dosage and fewer administrations2. SMA - Nusinersen (Spinraza) in clinical use enhance exon 7 inclusion in SMN2 to produce functional protein and lessen SMA caused by loss of SMN1. this results from blocking intron 7 splice site to promote exon 7 inclusion. 3. HD - IONIS-HTTRx in phase 3 trials - suppress translation of HTT mRNA containing CAG expansion. targets HTT-dependent SNPs so doesnt taget expansions in other genes.
what are the Challenges of using ASOs for the treatment of genetic diseases?
o Delivery to target tissueo Achieve sustained effect. Chemically stabilised forms of ASOs will require re-administration for most applications.o Difficult to achieve complete inhibition as there are large quantities of mRNA and lower levels of ASO within the cell.
describe CRISPR-Cas9 system and limitations
CRISPR-Cas9 is a highly cost-effective technique that allows specific targeting of gene manipulation via RNA-guided nuclease. once a cut is made, DNA with matching sticky ends can be incorporated by DNA repair mechanisms. Trials in b-thalassaemia, DMD, and freidreich ataxia. limitations:1. limited number of motifs to bind to in genome2. delivery to target cells3. although it has proofreading finction, some off target mutagenesis has been seen in similar sequences to target
describe small molecules for genetic treatment
- amnioglycosides promotes read-through of stop codons. amnioglycosides interact with A site of rRNA, altering conformation and reducing accuracy between mRNA-tRNA pairings allowing AA to be inserted instead of termination at stop codon. works best where low levels of functional protein can restore function. eg. Translana in DMD induces ribosomes to read through PTC. works on mRNA transcripts so patients with low mRNA levels may not respond. NICE approval 2. corrects folding/transport or activation of protein eg. Ivacaftor for CFTR to improve chloride channel transport (class III) or enhance folding (class II) eg. phe508del Lumacaftor.
Clinical features of HMSN1
distal muscle weaknesspes cavusfrequent trips, fallsdifficulty walking on uneven surfacesmuscle wastage in calves (inverted champagne bottle)
Molecular pathogenesis of PMP22 and disease
PMP22 expressed by Schwann cells of peripheral nerves - produces 2-5% of total myelinPMP22 dup causes increases PMP22 expression > disrupts regulation of myelin production > demyelination of peripheral nerves > abnormal axon function > reduced nerve conduction speeds and axonal lossPMP22 del > Reduced myelin > disruption of myelin junctions > impaired propogation of nerve potentials
What does HMSN stand for?
Hereditary motor and sensory neuropathy
Chromosome location for PMP22
17p12
Cause HNPP
85% caused by PMP22 deletions| 15% caused by LOF mutations in PMP22
Cause HMSN
???
Clinical features of HNPP
transient recurrent episodes of focal weakness and sensory loss- usually caused by pressure or injury
Why do PMP22 deletions and duplications occur?
PMP22 gene flanked by repeat regions; CMT1A-REP| NAHR between these regions leads to recurrent dels/dups
what are features of x-linked recessive inheritance?
- vertical transmission of carrier females to affected sons (50% chance)- all daughters of affected males have the pathogenic variant (obligate carriers)- daughters of female carriers have 50% chance of being a carrier- affected homozygous females are rare- affected males usually born to unaffected parents (may have affected male relatives)- no male to male transmission- pedigree mostly affected males, females are carriers only
why might females be affected by XLR disorder?
NAME?
Give examples of X linked disorders?
- DMD, BMD, -SBMA- androgen insensitivity syndrome- XLRP (retinitis pigmentosa)- haemophilia A and B - Christmas disease
what is DMD?
- 1/4000 births affected- rapidly progressive muscular weakness proximal > distal & lower> upper limbs with calf hypertophy- gower movement- early childhood age of onset & first signs are delayed milestones, delayed sitting and standing - cardiomyopathy >18 years, most common cause of death with respiratory complications- wheelchair bound by 12 - few survive beyond 30 yrs- males do not usually reproduce- full penetrance in males, females can be unaffected to severe (manifesting carrier)- >10x creatine kinase levels, less in females. CK is an enzyme released from muscle into the bloodstream following damage- out of frame deletions (be careful with duplications as dont always follow the rule)
what is BMD?
-1/18 000 male births- in-frame deletions in DMD- less severe & later onset- longer life expectancy (mid 40s)wheelchair bound >16- heart failure from DCM still common cause of death- females with DMD pathogenic variant at increased risk for DCM- preservation of neck flexor muscle strength (differs from DMD)- >5x creatine kinase levels , less in females
what is DMD-associated dilated cardiomyopathy (DCM) ?
- DCM between 20-40 years in males and later in females- usually no skeletal muscle disease- rapid progression to death in males and slower progression in females-increased creatine kinase levels , less in females
how do you test for DMD without genetics?
immuno staining shows lack of dystophin in skeletal muscle, cardiac and smooth muscle cells. Dystrophin plays a role in sarcolemma stability. In DMD it is absent but in BMD it is 20-100% - may be normal levels but reduced function
how do you genetically test for DMD?
- MLPA (80% mutations) - QF-PCR required or single del/dup or SNP array can offer higher resolution may be done solely by NGS in future- sequencing, NGS allows mosaics to be detected. sanger for familial mutations- RNA analysis deep intronic variants or complex rearrangements. usually muscle or urine but need to confirm in genomic DNA. can also confirm splicing outcomes and orientation of duplicationsTwo deletion hotspots: 30% exons 2-19 and 70% exons 45-55
What is traditional treatments for D/BMD (before gene therapies)?
steroids (improve muscle strength and motor function), physical therapy, anti-congestives and cardiac transplant in severe cases
what gene therapy is available for D/BMD?
- stop codon readthrough eg. translana. 15% of patients have PTC. Tretments allows alternative amino acids to be inserted at the site of the mutated stop codon & results in dystophin expression at 10-20% providing some function- exon skipping eg. exondys51 (80% of patients in theory). Interferes with splicing skipping the specific exon in DMD pre-mRNA to restore open reading frame and allow expression of shorter but functional protein
what is SBMA?
X linked disorder affecting males 1/300 000caused by CAG repeat in exon 1 of AR geneprogressive neuromuscular disorder with degeneration of motor neurons resulting in muscle weakness and muscle atrophy and reduced fertility (due to mild androgen insensitivity)GOF mutation. the more repeats there are the earlier the age of onsetonly occurs in Europeans or Asiansfemales not usually affected
what is androgen insensitivity syndrome (AIS)?
complete = total insensitivity to androgen and child develops female genitals. abnormal secondary sexual development at puberty and infertility in those with a 46, XY karyotypepartial = level of insensitivity determines how genitals develop (predominantly female, male or ambiguous)mild = typically male genitaliaAffects 2-5/100 000 who are genetically maleinfertility
What causes androgen insensitivity syndrome?
NAME?
WHat is haemophilia A and B (christmas disease)?
XLR disorders caused by mutations in F8 (A) and F9 (B) causing F8 and F9 to be inefficient at coagulation in the blood. Disorders are indistinguishable clinically. Clotting deficiency results in prolongued bleeding after injury. 10% of female carriers are at risk for bleeding. 1/6000 males have haem A and 1/30 000 males have haem B.
what is Fabry disease?
XLR lysosomal-storage disorder caused by mutations in GLA which encodes the galactosidase enzyme (breaks down a fatty substance). pathogenic variants prevent the enzyme from breaking down this substance leading to it damaging cells. symptoms include pain in hands and feet, inability to sweat, cloudiness in eye, angiokeratomas *dark red spots on skin), GI problems, tinnitus and hearing loss. life-threatening complications include kidney damage, heart attack and stroke. 1/5-10 000 affected. childhood onset. females may be affected
define anticipation?give examples
trinucleotide repeat expansions in successive generations in which the signs and symptoms of some genetic conditions tend to become more severe, more frequent or occur at an earlier age- these dynamic expansions are unstable and expand on transmission to next generation- eg. FRAX (XL) maternal CGG expansion- HD HTT CAG AD paternal- DM1 CTG DMPK maternal expansion
what causes trinucleotide repeats to expand?
replication slippage - mispairing, hairpin loops causing replication fork blockage, unequal crossing overresulting in one expanded and one contracted tract
define Age-related mosaicism?
Mosaicism is defined as the presence in an individual, or in a tissue, of two or more cell lines that differ in genetic constitution. As we age, somatic/germline mutations accumulate over the course of a person’s life resulting in age-related mosaicismeg. loss of X in males and females is normal age-related anaphase lageg. cancer is an example of age related mosaicism - mutations accumulate as we age
define Variable expressivitygive examples
degree to which phenotype is expressed varies between individuals with same genotypeeg. MArfan FBN1 - some are just tall and thin whilst others have heart conditionseg. NF1 - mildly affeected have cade au lait skin but more severely affected have neurofibromas. may also develop tumours. may also have ID, short stature or seizures.
define penetrancegive examples
proportion of individuals carrying a particular variant of a gene (allele or genotype) that also expresses an associated trait (phenotype)eg. BRCA1 - penetrance = 80% risk of BC. also affected by environmental modifiers such as smoking, diet, pregnancies etc
what is Age-related penetrance
penetrance is often expressed as a frequency, determined cumulatively, at different ages
define Sex-limitinggive an example
Sex-limited genes are genes which are present in both sexes but expressed only in one sex, and causes the two sexes to show different traits or phenotypeseg. Familial Male Precocious Puberty - males only have signs of puberty at 4 years
define epistasisgive an example
interaction between nonallelic genes in which one combination of such genes has a dominant effect over other combinations eg. Bombay phenotype
define pleiotrophygive an example
Pleiotropy occurs when one gene influences two or more seemingly unrelated phenotypic traits eg. usually metabolic pathway that affects different phenotypes such as PKU. depending on mutation involved may have reduced conversion of phenylalanine to tyrosine or ceased entirely. the failure to convert normal levels of phenylalanine to tyrosine results in less pigmentation being produced causing the fair hair and skin typically associated with phenylketonuria. also causes mental retardation and abnormal gait and posture.
what is antagonistic pleitropy?
the expression of a gene resulting in competing effects, some beneficial but others detrimental to the organism.Antagonistic pleiotropy hypothesis – some genes responsible for increased fitness in the younger, fertile organism contribute to decreased fitness later in life. An example is the p53 gene, which suppresses cancer, but also suppresses stem cells, which replenish worn-out tissue.
define: Amorphic, Hypomorphic Hypermorphic Antimorph and Neomorphic
Amorphic = LOFhypomorphic = partial loss through reduced function. usually recessive but occasionally dominant due to haploinsufficiency eg. Friedreich’s ataxia homozygous GAA repeat expansion. some cases are compound het with a point mutation. Homozygosity for inactivating mutations is embryonic lethal.hypermorphic = GOF - increased activity of normal functionantimorph = dominant negative (gene product adversely affects the normal, wild-type gene product)NEOMORPHIC - DOMINANT GAIN OF GENE FUNCTION that is different from normal function
what is a transition substitution?
a pyrimidine for a pyrimidine (C for T or vice versa) or a purine for a purine (A for G or vice versa)—
what is a transversion substitution?
Substitution of a pyrimidine by a purine or vice versa (C to an A)
define haploinsufficiency
A situation in which half amount of a gene product is not enough to maintain normal function for instance, individual with heterozygous mutation or hemizygous at a particular locus is clinically affected.
define linkage disequilibriumgive a CF example
: Linkage disequilibrium is the phenomenon by which there is a non-random association of alleles at two or more loci i.e. when variants co-occur together in an allele more than should be expected if random distribution of variants was occurring.Phe508del 98% occurs in cis with 9T - important if patient also has c.350G>A p.(Arg117His) and 5T (causes CF with 5T only)
give an example of XLD disorder?
Fragile X, - CGG trinucleotide repeat in FMR1X linked Alport syndrome - COL4A5. most common. kidney disease, hearing loss and eye abnormalities
give an example of an X-linked dominant disorder with male lethality
rett syndromeIntercontinentia pigmenti
what are the features of XLD inheritance?
- expressed in males and females- an affected female has a 50% chance of having an affected child- an affected male has affected daughters and unaffected sons- higher proportion of females affected than males- affected males have more severe phenotype - variability in females- may be mistaken for AD, unless there is an affected male who will have all affected daughters and no affected sons
When the mother is affected with an X-linked lethal disease what is the expected offspring ratio of M:F affected vs unaffected?
1/3 affected females, 1/3 unaffected females and 1/3 affected males
what are the features of an X-linked dominant disorder with male lethality in pedigrees?
- disorder observed exclusively in females- affected males rarely seen (except XXY)- history of miscarriages as 50% of males die- sex ratio of offspring is therefore skewed
describe X-linked male lethal Intercontinentia pigmenti
NAME?
describe X-linked male lethal rett?
NAME?
why is XLD Craniofrontonasal syndrome caused by variants in EFNB1 more severe in females?
-Cellular ‘interference’ between wild-type and mutant cell populations is the cause for the severe disease manifestation in CFNS females-males are asymptomatic/mild phenotype
why might an XL disorder present in a female?
- skewed x inactivation- variants in PAR regions are expressed on both X’s as escape X inactivation and can behave in AD manner eg. SHOX on PAR1- XLD- XO- deletion on one X
ADD TO CARDS what is a microdeletion? give examples of reciprocal, interstitial and terminal microdeletion syndromes.
<5-10 Mb, can be recurrent-monosomy and haploinsufficiency of dosage sensitive –genes. Also involves imprinted genes, unmasking recessive mutation, and positional effects- can result in clinically recognisable syndromecan be mendelian or contiguous eg. di george syndromefor every deletion there should be a duplication - reciprocal microdeletion/duplications from NAHR eg. HNPP/CMT1A 17p12 and Smith-Magenis syndrome (SMS)/Potocki-Lupski syndrome (PTLS) (17p11.2)Interstitial microdeletions: Di GEorge 22q11.2PWAS 15q11-q13NF1 17q11.2Miller-Dieker 17p13.3williams syndrome 7q11.23terminal microdeletions: wold-hirschorn del4ptercri du chat del 5pter
what is a microduplication
<5-10 Mb, can be recurrentregional trisomy and effects dosage sensitive geneseg. mendelian -PMP22 HNPP, - milder than deletions as cells more tolerant to gain than loss- FISH less sensitive for duplications-variable expressivity and reduced penetranceInterstitial eg. Duplication 7q11.23 (williams syndrome region duplication syndrome)
what causes microdeletions/microduplications?
- low copy repeats up to 0.5Mb that have high homology >97% and result in structural aberrations from NAHR. NAHR hotspots cause recurrent CNVs- may be interchromosomal- intrachromosomal (between 2 chromatids)- intrachromatid (within chromatid)- results in deletions, duplications, inversions and dicentric
what methods can be used to identify microdeletions and duplications?
NAME?
where is Charcot-Marie-Tooth (CMT1A)/Hereditary neuropathy with liability to pressure palsies (HNPP) located and what causes the syndromes? what are clinical features of the syndromes?
17p11.2- reciprocal 1.4Mb duplication/deletion including PMP22- caused by NAHR between two LCRs with 99% homologyCMT1A (80% of CMT1) = PMP22 duplications. CMT1 is a demyelinating peripheral neuropathy. distal muscle weakness and atrophy, slow nerve conduction. slowly progressive. foot drop, calf hypertrophy. HNPP - milder neuropathy. numbness, tingling and muscle weakness in the limbs. deletion of PMP22 (80%). other 20% have pathogenic mutation.
ADD TO CARDS where is smith magenis/Potocki-Lupski syndrome located and what causes the syndromes? what are clinical features of the syndromes?
17p11.23.7Mb recurrent del/dup generated by NAHR between LCRs. common del/dup occurs due to NAHR between proximal and distal low copy repeats. smith-magenis syndrome = developmental delay, hypotonia and distinctive facial features. caused by de novo 17p11.2 deletions. (70%)potocki-lupski syndrome - dev delay, autism and hypotonia. de novo duplications of 17p11.2
what causes Prader-Willi syndrome (PWS)/Angelman syndrome (AS) del 15q11-q13 and what are clinical features?
PWS = hypotonia, feeding difficulties, obesity, dev delay, insatiable appetite. - paternal contiguous gene deletion resulting from NAHR between low copy repeats (75%) of 15q11q13 leading to absence of expression of paternally-imprinted genes. also caused by mat UPD and IC deletion. there are 4 large clusters of complex repeats termed BP1-4, with common deletion ranging from BP1 or 2 to BP3. SNURF-SNRPN, MAGEL2, C15orf2 involved.angelmann = severe developmental delay/intellectual disability, severe speech impairment, gait ataxia , happy demeanor, microcephaly, sezures. Disruption of UBE3A in 15q11q13. 70% have maternal deletion.
what causes Neurofibromatosis type 1 (NF1)? where is the location? what are clinical features?
del 17q11.2multiple café-au-lait spots, neurofibromata, learning difficultiesheterozygous loss of function mutations in the NF1 gene at 17q11.2 as well as by an LCR-mediated 1.5Mb deletion that encompasses NF1 and other flanking genes and pseudogenes
what is the location of Wolf-Hirschhorn syndrome , what causes the syndrome and what are the clinical features?
del 4ptercraniofacial features (greek warrior helmet appearancedev delay, growth delay, ID, seizurescontiguous gene deletion up tp 60% de novo and 40% have unbalanced translocation with deletion of 4p and partial trisomy of different chromosome arm. may require FISH for detectionCandidate genes that may contribute to the WHS phenotype include WHSC1, LETM1 (seizures) and FGFRL1 (craniofacial phenotype).
what is the location of cri du chat syndrome , what causes the syndrome and what are the clinical features?
del 5ptercat-like crysevere dev delay/IDmicrocephaly, hypotonia, hyperterlorism, variable seized deletions of 5p from 5p13-5p15size of del correlates with phenotype85% are de novo deletion and 15% are unbalanced translocationCTNND2 causes ID
define haploinsufficiency?
both copies of a gene need to be functional in order to express the wildtype. 50% of the normal gene product/expression/activity is not enough. Inactivation/loss of a single allele (leaving the second allele unaffected) produces a clinical phenotypeA gene is likely to be haploinsufficient if all mutation types (missense, nonsense, gene deletion etc.) produce the same phenotype
give an example of a haploinsufficient single gene?
PMP22 - HNPP 80% caused by deletion of a ~1.5Mb region at 17p11.2 and 20% caused by mutationrepeated focal pressure neuropathies HNPP is haploinsufficient whilst PMP22 dup CMT1A is GOF
give an example of a haploinsufficient contiguous gene deletion syndrome?
22q11.2 Di George syndrome - tbx1 HI causes heart defects and dysmorphology
give example of HI cancer gene?
TP53 - Thought to be typical two-hit model BUT LFS tumours analysed for LOH (loss-of-heterozygosity) show it occurs in ~60%, so ~40% of tumours have a presumably functioning second copy
what is a gain of function mutation?what is a hypermorph and a neomorph?
- increase in gene expression or product develops new functionÔÉò Hypermorph: an allele that produces an increase in quantity or activity of its productÔÉò Neomorph: an allele with a novel activity or product.
give examples of GOF mutations?
HD = CAG TNR exon 1 HTT polyglutamine tract 36 repeats or more. acquires novel deleterious function - deletions of region do not cause disease so GOF. forms inclusion bodies containing huntingtin which forms abnormal B sheetsDM1 - CTG toxic gain of function in DM1 and CCTG in DM2Achondroplasia - inherited short stature FGFR3 tyrosine kinase receptor which negatively regulates bone growth. mutations activate the receptor, limiting bone growth. Gly380Arg) accounts for >99% of cases. high de novo mutation rate. FGFR3 mutations also cause thanatophoric dysplasia TD1 and 2, hypochondroplasiaBCR-ABL1 fusion gene in CML: t(9;22)(q34;q11) - novel tyrosine kinase. imatinib is TKI. MRD by RT-PCR quantifies levels of BCR-ABL1 mRNA transcripts in blood and bone marrow samples. determines treatment response. CMT PMP22 - 1.5Mb duplication 17p11.2 - 80% of CMT1 cases. PMP22 codes for peripheral myelin protein. duplication generated by NAHR between sequences that flank the gene. reciprocal deletion causes HNPP
what is a dominant negative mutation?
mutations that reduce the function of the protein encoded by the normal copy of the gene- only seen in heterozygotes and cause more severe effect than no gene product- <50% residual function-
give examples of dominant negative mutations
osteogenesis imperfecta- COL1A1 or COL1A2 causes 90% of cases- Haploinsufficiency (type 1): null variants and NMD results in milder phenotype as amount of collagen produced is reduced- Dominant negative (Type 2): 80% caused by replacement of Glycine in Gly-X-Y in triple helix domain. disripts formation of triple helix and causes severe diseaseMarfan - FBN1- haploinsufficiency or dominant negative- dominant negative: usually cysteine substitutions Haploinsufficiency - nonsense/FS lead to NMD and decreased amount of fibrillin and aortic wall strength
which classes of mutations are pancreatic insufficiency associated with?
class 1, 2 and 3levels of pancreatic function correlate well with CFTR genotype.
what is the Significance of the intron 8 poly(T) and poly TG tract?
- exon 9 is 90% skipped when the length of the tract is reduced to 5T- The pathogenicity of the poly(T) tract is also mediated by the length of the adjacent poly(TG) tract, where long TG tracts are more likely associated with disease phenotype than shorter tracts (higher penetrance). Common TG lengths = 11, 12 or 13.- Increased skipping of exon 9 is caused by TG12, TG13 and 5T
what is the significance of 5T in cis with p.(Arg117His) ? what about 5T in trans with a CF mutation or 5T/5T?
- modifies the expression- 5T/Arg117His hets together with a typical severe pathogenic variant can cause classical CF of variable severity- 7T/Arg117His hets (most 7T associated with TG11) and a typical severe mutation result in much more variable phenotype and can even be benign- 9T/Arg117His hets are very rare and thought to be benign- highly variable and more likely CFTR related disease eg. CBAVD, bronchiectasis/pancreatitis
which variant is 9T seen exclusively in cis with?
phe508del
when is reflex testing for 5T recommended?
NAME?
what treatments are available for CF patients?
NAME?
why might there be elevated neonatal IRT apart from CF?
neonatal stress (low Apgars), respiratory distress, hypoglycaemia, or serious congenital abnormalities such as trisomies 13 and 18PPV is low for IRT
describe NBS for PKU?
NAME?
describe NBS for sickle cell disease?
- sCD means cells are unable to deform as they pass through narrow capillaries- serious infection, chronic pain, pulmonary hypertension, proliferative retinopathy, organ damage, aplastic crisis, stroke or even death- haemaglobin A accounts for 95% normal haemoglobin and accounts for two alpha and two beta chains. SCD is caused by mutation in B haemoglobin gene HBB. - more common in people from tropical and/or sub-tropical regions where malaria was common as carrying a single sickle cell gene conferred an advantage- screen positive = immunisations and antibiotics- screened using HPLC, isoelectric focussing and capillary electrophoresis- caused by HBB gene, c.20A>T p.Glu6Val detected by ARMS or pyrosequencing- HBB, HBA1 and HBA2 genes can be tested for thalessaemia
describe MCADD NBS?
- problems breaking down fats leading to illness or death- AR ACADM - Single common mutation c.985A>G (p.Lys304Glu) is responsible for 88% - screen positive infants are put on special diet- mass spec of C8 levels relative to c10- targeted testing of the c.985A>G followed by extended screening
what is the MOI and mutation for FRAX?what FMR1-related disorders are there?
X linked dominant, variable penetrance, 5’ UTR CGG repeat >200 causes methylation gene silencing- small proportion of cases due to point mutations or partial or whole gene deletions or duplicationsfragile X tremor ataxia syndrome/ primary ovarian syndrome, fragile X syndrome
give examples of genes that cause several diseases (clinical heterogeneity)
- PMP22 17p11.2= peripheral myelin protein 221.5 Mb deletion causes HNPP Hereditary Neuropathy with Liability to Pressure Palsies. muscle weakness and atrophy. CMT1A = PMP22 duplication 1.5Mb duplication/GOF mutation. progressive muscle weakness. - FGFR3 - tyrosine kinase receptor that negatively regulates bone growth. GOF mutations cause hypochondroplasia, achondroplasia, TD, Muenke Syndrome. somatic activating mutations cause bladder cancer. - AR gene. polyglutamine expansion causes SBMA. muscle weakness. onset decreases as expands. het females usually unaffected. androgen-insensitivity syndrome- complete, partial and mild
give examples of diseases caused by several genes? (genetic heterogeneity)
cardiomyopathy - shortness of breath, chest pain progressive heart failure to sudden cardiac deathMost common genes mutated= MYH7 (40%), MYBPC3 (40%), TNNT2 (5%), TNNI3(5%), TPM1 (5%)Incomplete penetrance and variable expressivityCaused by dominant negative, haploinsufficiency and GOF mutations
what is Pleiotropy
occurs when one gene influences two or more seemingly unrelated phenotypic traits
what is a polygenic risk score?
quantify cumulative effects of no. of genes, which may individually only have small effect on susceptibility; score reflects sum of all known risk alleles, weighted by how risky each variant known to bepredict persons likelihood of displaying a trait• Genome-wide data allows access to millions of common genetic variations associated with these conditions; risk score for each person calculated with aim to inform clinical management
what are the benefits of polygenic risk scores?
• Helpful in targeting people at higher-risk of conditions where increased surveillance + preventative treatment/surgery available• Help to match drugs in clinical trials to individuals who are most likely to benefit from them- personalize preventative measures
what are the drawbacks of polygenic risk scores?
• Not diagnostic: high risk score doesn’t mean person will definitely develop condition + vice versa• Results need to be communicated carefully to minimise risk of confusion• Requires consent for genetic testing; may be difficult to obtain• Accuracy; majority calculated from European DNA sequences; less accurate for other populations
why are repeat expansions known as dynamic? what causes changes in TNR numbers?
premutations: can expand or contract when transmittedDynamic mutations also show somatic instability, which accounts for some of the phenotypic variability.strand slippage during DNA replication in actively dividing cells
what mutation causes Motor neurone disease (MND) and frontotemporal dementia (FTD) ?
AD c9orf72 hexanucleotide repeat GGGGCC expansion
what mutation causes DM2? what are symptoms? what is the repeat size range?
tetranucleotide repeat CCTG in intron 1 ZNF9muscle weakness, muscle pain, Full penetrance, mutation alleles >75 repeats, up to 11,000 repeats
give examples of triplet repeat disorders caused by LOF mutation?
Fragile X Syndrome (both FRAXA and FRAXE) and Friedreich Ataxia (FRDA)expansions result in loss of gene product or functionPoint mutations are rare in affected patients (<1% Fragile X cases, ~2% FRDA cases)
describe freidrich ataxia, what the disease mechanism and mutation is, clinical features
- AR so anticipation not observed, carriers unaffected- most common form of ataxia with 1/50 carrier frequency- progressive ataxia (co-ordination, balance and speech) muscle weakness and HCM- homozygous expansion of a polyE glutamic acid GAA repeat in intron 1 of frataxin (FXN)- 2% of patients have expansion and LOF mutation- Nonsense, missense, frameshift, and splicing defect in trans- mutation causes defective transcription of the FXN gene, leading to deficiency of frataxin (mitochondrial protein)-PCR, TP-PCR and southern blot can be used to test & MLPA + sequencing if strong clinical suspicion and only one expansion identified
give examples of Coding GOF polyglutamine disorders (CAG repeat expansions) ?
SBMA, HD, DRLPA, SCAs
give examples of non coding GOF disorders?
DM1, DM2, SCA 8 + SCA 12
what mutation causes DM1? what are clinical features?what are the repeat sizes?
-GoF - Haploinsufficiency caused by CTG expansion-RNA GoF- CUG expansions in 3’ UTR fold into RNA hairpins that accumulate in ribonuclear foci-encoding DMPK protein ‚Äì serine-threonine kinase, mainly expressed in heart + skeletal musclemuscle weakness + wasting ,cataracts, cardiac defects, floppy baby + respiratory distressnormal = up to 36 repeats (same as HD)premutation 37-50full penetrance alleles 51-150 juvenile > 150congenital = >2000
what is the mutation that causes HD? what are the repeat ranges? what are clinical symptoms? what testing can be offered to parents who do not want to know disease status?
CAG expansion in exon 1 HTT, higher repeats = earlier onset<27 = normal27-35 = intermediate36-39 = reduced penetrance (some elderly unaffecteds)>39 = expansion>60 = juvenile- chorea, dystonia (muscle spasm), psychiatric disturbance, involuntary movements, cognitive decline- large expansions exclusively paternal- exclusion testing can be offered where parent doesn’t want to know status (25% risk to child)
what therapies are in development for TNR disorders?
- Antisense oligonucleotides- IONIS-HTT: nhibit expanded mRNA to reduce concentration of mutant HTT protein2. RNAi- mouse model of SCA1, with adeno-associated virus (AAV) vectors expressing short hairpin RNAs that efficiently reduced expression levels of mutant protein3. Protein aggregation inhibitors e.g. (2)-Epigallocatechin-3-Gallate (EGCG) for HD
describe clinical features of FSHD, the mutation responsible and how to test?
FSHD - Facioscapulohumeral muscular dystrophy- 95% of cases are FSHD1- muscle weakness- FSHD1 caused by AD contraction of D4Z4 repeats (contains DUX4 gene)<10 repeats = pathogenic- results in reduced transcription of DUX4- diagnosed by southern blot due to large size using restriction enzymes- FSHD2 caused by SMCHD1 mutations and diagnosed by bisulphite PCR and NGS
give examples of poly-alanine expansion disorders?
XLMR - x-linked mental retardation ARX geneHand–foot–genital syndrome - HOXA13
List three differences between the nuclear and mitochondrial genomes
mitochondrial genome is circular and double-strandedmitochondrial genome contains 37 genesThere are no introns - only 3% is non-codingmitochondrial mode of inheritance is strictly maternalEach cell can contain 100 to 10,000 copies of the mt genome but only one copy of nuclear
define homoplasmy, heteroplasmy and mt genetic bottleneckwhat is the threshold effect?
mtDNA can all be identical (homoplasmy)mixture of two or more mt genotypes (heteroplasmy)The percentage level of mutant mtDNA may vary among individuals within the same family, and also among organs and tissues within the same individual (mt genetic bottleneck).• The threshold effect is that there is a level of variant mtDNA that can be tolerated by the cell, however above a certain level (which is tissue specific) oxidative phosphorylation cannot be maintained and disease manifests
Describe 3 considerations for interpretation of pathogenicity unique to mtDNA variants
Homoplasmic variants inherited from a homoplasmic clinically unaffected mother are unlikely to be pathogenic.mitomap - phenotypes and genbank frequencyThe level of heteroplasmy must be accurately determined and interpreted in the context of the tissueEvolutionary conservation and functionality eg. MitoTIP
why is mitochondrial genetic testing particularly difficult?
- variant differs by tissue type and age of patient- Some pathogenic variants (e.g. m.3243A>G) are lost from tissues such as blood that undergo rapid mitotic division- a neg result doesnt mean the patient doesnt have variant- DNA extracted from the affected tissue is preferred - Genotype-phenotype correlations are poor
Clinicians have referred an adult presenting with optic neuropathy to the highly specialised mitochondrial diagnostic service. Describe the appropriate testing pathway and any relevant candidate genes and variants for targeted analysis
MT-ND1 m.3460G>A , MT-ND4 m.11778G>A and MT-ND6 m.14484T>C targeted testing of 3 common mutations eg. pyrosequencingmitochondrial WGSDNAJC30 single gene sequencingreferred from Consultant Ophthalmologist, Neurologist or Clinical Geneticistclinical assessment, family history, biochemical testing, histopathological examination (muscle biopsy) and direct molecular testing
Name the gene responsible for encoding mitochondrial DNA polymerase
POLG
what is MELAS? what is the mutation? what sample types should be used?
Mitochondrial encephalopathy, lactic acidosis and stroke-like episodesMT-TL1 m.3243A>G urine or muscle best samples
ADD TO CARDS what is leigh syndrome? what is the mutation?
MT-ATP6 m.8993T>C (also MT-ND1, 4 and 6)Encephalopathy, lactic acidosis
what techniques are used for mt-DNA testing? what is the best sample for testing?
common mutations = sequencing/pyrosequencingrearrangements such as deletions = long range PCR. multiple mtDNA deletions may be due to nuclear gene mutation such as POLG for exampleRT-PCR - copy number and WGS if increased copy number as likely to be a mtDNA mutationif not mt DNA gene need to consider nuclear sequencing - encodes mt subunits and mt genome maintenancemuscle is the best tissue to test
why is mtDNA testing particularly difficult for prenatal diagnosis? what methods can be undertaken to prevent mt disease transmission?
- heteroplasmy difficult to interpret- % heteroplasmy in CVS may not represent other tissues and levels may change during development- difficult to predict severity of disease- ooctye donation or nuclear transfer where the nuclear genome from the oocyte or embryo of an affected woman is transplanted into a donor enucleated oocyte or embryo with healthy mt. nuclear DNA can be transferred between unfertilised oocytes using polar body transfer or maternal spindle transfer or between fertilised zygotes using pronuclear transfer
what is a chromosome instability/breakage syndrome?
inability to repair a particular type of DNA damageusually ARhypersensitive to certain drugs leading to chromosomal rearrangementsincreased predisposition to cancerdiagnosed through cytogenetics biochemical and molecular methods
Fanconi Anaemia (FA) - what are the genes responsible, clinical features and testing?
most common breakage syndromeFANCA is the most common cause, but 15 different groupsmostly AR, but also XL (FANCB - males affected) and ADgrowth retardation, dev delay, skeletal malformations, increased susceptibility to leukaemia, anaemiaMutated cells have deficient ability to excise UV-induced pyrimidine dimers from the cellular DNA - it leads to double-strand breaks in the S phase of the cell cycleTesting: culture cells with cisplatin (interstrand cross-linking agent) causes breakage and sequence FA genes
Bloom syndrome-what is the gene responsible, clinical features and testing?
AR BLM gene - DNA helicase tumour suppressor gene that suppresses inappropriate recombinationsun sensitive rash, growth deficiency, immunodeficiency, predisposition to cancer, infertility in malesincreased SCE levelgene sequencing and count SCE frequency in 20 metaphases
Ataxia Telangiectasia (AT) - what is the gene responsible, clinical features and testing?chromosomal breakage
AR disorderataxia-jerky movements, immunodeficiency, cancer predisposition - leukaemiasATM gene sequencing - usually compound het mutations but also some dominant negative heterozygous missense variants. Gene codes for a kinase which signals ds breaks and nonhomologous exchange. cytogenetics - radiation causes chromosome breakage
Xeroderma Pigmentosum - what is the gene responsible, clinical features and testing?
sun sensitivity, cancer predisposition, hearing loss, cognitive impairment, genetic sequencing- 8 genes involved in nucleotide excision repeair (NER) eg. XPC, ERCC2 and POLHcaused by UV radiation damaging genes that control growth and division - cells die or grow uncontrollably leading to cancers
why might there be an abnormal phenotype in a patient with a balanced rearrangement? (8 answers)
- gene disruption (most common cause) - truncated or no protein produced. may unmask recessive condition if SNV in other allele. FISH can detect this and NGS with paired end reads. can help to refine breakpoints. can create fusion genes.2. cyrptic imbalance - more complex (>3 breaks the more likely to have cryptic imbalance). may have dels, ins or inv near breakpoint or not at breakpoint site. may be inherited or de novo. phenotype depends on size of cryptic imbalance. 3) position effect - moves gene away from cis elements such as enhancer, inhibitor or closer to enhancer of another gene causing cancer, regulatory element may be moved closer to another gene altering expression levels and euchromatic material may be moved to heterochromatic region causing silencing and vice versa. 4) imprinting disturbance - imprinted region moved away from IC5) UPD - robertsonian and translocation carriers may have offspring with UPD. relevant if genes are involved in imprinting or unmasks recessive allele eg. isodisomy. very rare6) X;autosome rearrangements - males always infertile. due to spermatogenic arrest. Females - if normal X inactivated it is balanced. If abnormal X with XIC inactivated it will result in some autosome being inactivated too and translocated X will not be inactivated causing abnormal dosage. also if derivative autosome contains XIC it will also be unbalanced. t(Y;autosome) carriers usually infertile due to spermatogenic arrest. 7) mosaicism - may be mosaic for unbalanced karyotype in certain tissues Eg Pallister-Killian - normal in blood. or may have interchange trisomy followed byl loss of normal chromosome to give balanced karyotype in blood8) may be IF and cause is due to something else
why does a balanced translocation result in infertility?
disrupt gamete formation especially in malesfailure of pairing of homologous regions in the quadrivalent formed at meiosis I by a reciprocal translocation, which then interfere with the X-Y bivalent disrupting meiosis.robertsonian translocation - p arms interfere with X;Y bivalentfertility may vary between males in same family
what proportion of moderate to severe ID is genetics, environment and unknown? what % of population have ID? why is a diagnosis useful?
up to 40% geneticup to 30% environmentalrest unknown1-3%prognosis, clinical care and educational needs, support groups and prenatal diagnosis. currently incurable
how does array CGH compare to karyotype for ab USS findings?
Increases detection of chromosome abnormalities by up to 6% compared to karyotype for USS findings
what is the diagnostic yield of exome sequencing for patients with severe ID
25% - usually done after a normal array or WGS
how does use of array as first line test for DD, LD, autistic spectrum disorders or multiple congenital anomalies improve detection rate compared to karyotype?
use of arrays increased the detection rate from approx 3% with karyotyping to approx 15-20% and represent a 100 fold higher resolution approach
what is a marker chromosome?AKA: Small supernumerary marker chromosome (sSMC)/ Extra structurally abnormal chromosome (ESACs)/Accessory chromosomes
structurally abnormal chromosome that cannot be identified by conventional banding alone and is equal to or smaller in size than chromosome 20 of the same metaphase spreadmajority derived from acrocentric chromosomesCan be inverted duplication, ring, centric (have centromere), neocentric (new centromere)some produce abnormal phenotype but more than half don’t- clinical outcome difficult to predict - especially prenatally. If identifyable use der(chr) instead of marker50% are mosaic - almost all pallister-killian are mosaicmay cause infertility by interfering with meiosis80% are de novo - usually abnormal phenotype & low recurrence risk<0.1% of live births23% inherited but most without clinical signs (mostly maternal)‚Ä¢ Very small markers are prone to loss during cell division ‚Äì mosaicism often seen
